Prior References--GRF
Human Growth hormone-releasing Factor (hGRF) is a 44 amino acid peptide having growth hormone (GH) releasing activity as reported by Guillemin et al., 218, Science 585 (1982). hGRF is usually isolated from pancreatic human tumor cells (hpGRF). hpGRF has the structure H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser- Ala -Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-A rg-Gly-Ala-Arg-Ala-Arg-Leu-NH2. Since the initial discovery of hGRF, several peptides, designated herein as analogs, having deleted, substituted or otherwise modified sequences have been reported to have GH releasing activity Rivier et al., 300 Nature 276 (1982) reported a peptide that terminates as a free carboxycylic acid and differs from hGRF by the absence of the C-terminal tetrapeptide amide -Arg-Ala-Arg-Leu-NH.sub.2. Rivier et al. tested a number of shortened GRF analogs and reported that, when compared in vitro with the parent hpGRF(1-40)-OH, the
following analogs exhibited similar activity: hpGRF(1-29)-NH.sub.2, hpGRF(1-32)--NH.sub.2, hpGRF(1-39)--NH.sub.2, hpGRF(1-40)-NH.sub.2, and hpGRF(1-27)-NH.sub.2.
Numerous synthetic GRF peptides and GRF analogs have been patented U.S Pat. No. 4,610,976 to Bohlen discloses 44 amino acid synthetic peptides described as extremely potent in stimulating the release of pituitary GH in mammals. These synthetic peptides, biologically active fragments thereof, analogs thereof, or nontoxic salts thereof can be dispersed in a pharmaceutically acceptable carrier and administered for diagnostic or therapeutic purposes. The 44 amino acid polypeptide is believed to be porcine GRF. U.S. Pat. No. 4,605,643 to Bohlen discloses a 44 amino acid synthetic polypeptide that is the replicate of the native GRF of the sheep hypothalamus. The peptide is extremely potent in stimulating the release of GH in mammals. The patent states that the peptide, biologically active fragments thereof, analogs thereof, or nontoxic salts thereof may be administered to animals for therapeutic or diagnostic purposes. As examples of biologically active fragments, Bohlen states that fragments 34-43 residues in length, or even shorter fragments, e.g. oGRF (1-32), that retain an --OH or --NH.sub.2 of the C-terminal and retain the desired biological activity are suitable. U.S. Pat. No. 4,595,676 to Spiess discloses the synthesis of rat hypothalamic GRF. A number of polypeptides which have 44 amino acids and are useful in stimulating the release of GH in animals are disclosed. Reference also is made to biologically active fragments of the polypeptides. U.S. Pat. No. 4,585,756 to Brazeau discloses a 44-residue polypeptide isolated from purified extracts of bovine hypothalami and useful for promoting the growth of animals. Reference is made to biologically active fragments thereof, including bGRF(1-40) and bGRF (1-37) or shorter fragments. U.S. Pat. No. 4,563,352 to Rivier describes the synthesis of human pancreatic GRF and biologically active fragments thereof and provides synthetic peptides useful in stimulating the release of pituitary GH in mammals. U.S. Pat. No. 4,562,175 to Chang discloses a synthetic peptide GRF useful in growth inducing pharmaceutical compositions. The peptide is based on the structure of human pancreatic GRF. The synthetic peptide differs from the natural peptide in that norleucine is substituted for methionine at position 27. Other analogous peptides susceptible to a similar modification also are disclosed, including [D-Ala.sup.2, Nle.sup.27 ]-hpGRF-(1-44)--NH.sub.2 and [D-Ala.sup.2, Nle.sup.27 ]-ratGRF(1-43)--NH.sub.2). U.S. Pat. No. 4,529,595 to Rivier discloses analogs of hpGRF useful in stimulating the release of pituitary GH in mammals. Biologically active fragments, said to generally extend from the N-terminal to a residue between positions 27 and 32, also are disclosed. U.S. Pat. No. 4,528,190 to Vale provides synthetic polypeptides, useful in stimulating the release of pituitary GH in animals, which have resistance to enzymatic degradation in the body. U.S. Pat. No. 4,518,586 to Rivier describes a 44-amino acid synthetic polypeptide in which any or all of the residues between the 29th and 44th residues may be deleted. U.S. Pat. No. 4,517,181 to Ling discloses synthetic porcine GRF peptides which promote the release of GH by the pituitary gland and teaches that deletions can be made beginning at the carboxyl end of the peptide to create fragments that retain substantial portions of the potency of the peptide. U.S. Pat. No. 4,617,149 discloses a class of 44-amino acid polypeptide analogs of hpGRF bearing substitutions of the amino acid at position 27. Other similar GRF analogs are disclosed in U.S. Pat. Nos. 4,622,312 and 4,626,523.
Similarly, there have been many publications relating to GRF analogs: Ling et al.. Synthesis and In Vitro Bioactivity of C-Terminal Deleted Analogs of Human Growth Hormone-Releasing Factor, Biochem. Biophys. Res. Commun., 123(2), 854-861 (1984); Ling et al., Synthesis and In Vitro Bioactivity Human Growth Hormone-Releasing Factor Analogs Substituted at Position-1, Biochem. Biophys. Res. Commun., 122(1), 304-310 (1984); Wehrenberg et al., In Vitro Biological Potency of Rat and Fragments, Biochem. Biophys. Res. Commun., 115(2), 525-530 (1984); and Lance et al., Super-Active Analogs (1-29)-Amide, Biochem. Biophys. Res. Commun.. 119(1), 265-272 (1984).
2. Prior References--N-Terminal Modified GRF
Modification of the N-terminal end of GRF and its analogs is known in the art. Hodate et al., Endocrinol. Japan. 33:519 (1986) discloses a GRF analog formed by a D-Tyr substitution at the N-Terminal Tyr of native GRF. Frohman et al., J. Clin. Invest. 78:906 (1986) discloses GRF analogs which have modifications to the N-terminal end. Frohman suggests that the more active analogs have modifications which increase the protein's resistance to enzymatic degradation at the N-terminus. Similarly, U.S. Pat. No. 4,659,693 discloses a GRF analog in which an N,N'-dialkyl substituted guanidino amino acyl residue is used at position 1 or 2 of the protein.
All the N-terminal modifications in the prior references have been directed to methods for increasing the protein's resistance to enzymatic digestion; the modified proteins are designed to be poor substrates for aminopeptidases and dipeptidylaminopeptidases which would digest the peptide. Such modifications have involved adding D amino acids or chemically modifying the N-terminal end of the protein. None of the prior references discloses adding natural amino acids such as proline to the N-terminal of the protein; such an addition would not be expected to increase resistance to enzymatic attack.
Although numerous patents and publications relating to GRF analogs have been disclosed in the prior art, there exists a continuing need for synthetic GRF analogs which stimulate the release of GH and induce the beneficial effects associated therewith, particularly GRF analogs which are more potent than native GRF.